Paving the way towards effective plant-based inhibitors of hyaluronidase and tyrosinase: a critical review on a structure–activity relationship

Abstract Human has used plants to treat many civilisation diseases for thousands of years. Examples include reserpine (hypertension therapy), digoxin (myocardial diseases), vinblastine and vincristine (cancers), and opioids (palliative treatment). Plants are a rich source of natural metabolites with multiple biological activities, and the use of modern approaches and tools allowed finally for more effective bioprospecting. The new phytochemicals are hyaluronidase (Hyal) inhibitors, which could serve as anti-cancer drugs, male contraceptives, and an antidote against venoms. In turn, tyrosinase inhibitors can be used in cosmetics/pharmaceuticals as whitening agents and to treat skin pigmentation disorders. However, the activity of these inhibitors is stricte dependent on their structure and the presence of the chemical groups, e.g. carbonyl or hydroxyl. This review aims to provide comprehensive and in-depth evidence related to the anti-tyrosinase and anti-Hyal activity of phytochemicals as well as confirming their efficiency and future perspectives.


Introduction
Before the advancement of modern science, herbal medicines were widely used in ethnomedicine. Modern medicine primarily uses plant active ingredients that can be divided into primary and secondary metabolites. Secondary metabolites are defined as substances that are not directly necessary for the organism's growth and development. It is believed that they play an important role in adapting plants to changing external conditions. Secondary metabolites are formed in various metabolic pathways, including amino acids and sugars intermediates, therefore, plants can synthesise many structurally diverse metabolites. It is estimated that plants produce at least 250,000 natural products, many of which have not yet been identified and characterised in structure and biological activity 1 . The enormous diversity in the plant world (250,000-300,000 species) has provided many substances currently used in treatment 2,3 . The isolation of morphine and codeine from Papaver somniferum L. ensured an effective pain treatment 4 . Obtaining digoxin from Digitalis purpurea L. allowed for an effective treatment of atrial fibrillation and heart failure 5 . Salicylic acid, which is found, among others, in Salix alba L., after esterification forms acetylsalicylic acid, used in pain and thrombotic diseases 6 . The isolated artimizin from Artemisia annua L. is used in the treatment of a resistant form of malaria 7 . Other compounds used in the treatment are capsaicin from Capsicum annuum L. 8 , quinine from Cinchona L. 9 , and inulin 10 . Additionally, some compounds, after structure modification, have contributed to a new group of drugs. A good example is a phlorizin, which has served as the host structure for SGLT2 sodium-glucose co-transporter inhibitors, e.g. dapagliflozin 11 . The development of new branches of science, such as genetics, biotechnology, and molecular biology, allowed for a better understanding of diseases' pathophysiology 12,13 . Hyals and tyrosinases are an underestimated group of enzymes commonly found in the world of organisms. Hyal, due to its participation in many biological processes, such as fertilisation, diffusion of toxins and microorganisms, inflammatory and allergic reactions, and cancer development, is perceived as a potential therapeutic target 14 . Besides, the inhibitors of tyrosinases and Hyals can be used in cosmetology to develop cosmetics or drugs used in dermatological diseases, such as eczema, acne, discolouration, and photoaging [15][16][17] .
This study aims to systematise knowledge about the Hyal and tyrosinase inhibitors. We have hypothesised that there are the plant-based compounds and their chemically modified derivatives which inhibit the Hyal and tyrosinase.

Types of inhibition
Inhibition is the process that slows down or completely stops a chemical reaction by a substance called an inhibitor. Depending on the way of binding of the inhibitor with the enzyme, one can distinguish reversible and irreversible inhibition (Table 1).

Irreversible inhibition
An irreversible inhibitor is a compound with a structure similar to the substrate or product that forms a covalent bond with a group present in the active centre. In the case of irreversible inhibition, it is not necessary to maintain the inhibitor concentration at a sufficient level to ensure enzyme-substrate interaction. The complex formed does not dissociate, so the enzyme is inactive even when the inhibitor is absent. In contrast to irreversible inhibition, reversible inhibition is characterised by dissociating the enzyme-inhibitor complex. There are the following types of reversible inhibition: competent, incompetent, acompetent, and mixed 18 .

Competent inhibition
The inhibitor shows a structural similarity to the substrate with which it competes for access to the enzyme's active centre. The enzyme-inhibitor complex converts to an enzyme-substrate complex, and the inhibitor is displaced from the enzyme's active site. The degree of inhibition of the enzyme depends on the concentration of the substrate. The higher substrate concentration relative to the inhibitor, the fewer enzyme molecules will bind to the inhibitor. Therefore, the inhibition of the reaction caused by a competent inhibitor can be reversed by increasing the substrate concentration.

Noncompetent inhibition
A noncompetent inhibitor usually bears no resemblance to the structure of the substrate. The inhibitor binds to the enzyme at a different site than the substrate. The inhibitor can attach to the free enzyme or the enzyme-substrate complex. Unlike competent inhibition, incompetent inhibition does not depend on the concentration of the substrate.

Accompetent inhibition
The inhibitor binds to the enzyme-substrate complex, forming an enzyme-inhibitor-substrate complex.

Mixed inhibition
It is a case of partially competent and incompetent inhibition. The inhibitor can bind to both the free enzyme and the ES complex, reducing the maximum rate 19 .

Hyaluronic acid and hyaluronidase
Hyal is responsible for the hydrolysis of hyaluronic acid (HA). HA is a linear, high molecular weight unsulfated polysaccharide composed of alternating N-acetyl D-glucosamine and D-glucuronic acid residues linked by glycosidic bonds. That compound is present both in prokaryotes and eukaryotes, commonly found in many tissues and body fluids, such as muscles, joints, synovial fluid, skin, and vitreous body. In addition to its structural functions, it is involved in wound healing, inflammation, and tumour development. Hyals are a group of enzymes commonly found in nature, e.g. they are an important component of the venom of bees, spiders, and snakes [20][21][22] . Based on the structure and mechanism of action, there are three classes of Hyals ( Figure 1). The first class includes endo-b-N-acetylhexosaminidases (EC 3.2.1. 35) present in mammals and in snake and hymenoptera venom. They hydrolyse b-1,4-glycosidic bonds in HA. The second class is endob-D-glucuronidases (EC 3.2.1.36), which hydrolyse the b-1,3-glycosidic linkages in the HA and are present in invertebrates. As a result of the activity of both enzymes, tetra, and hexasaccharide of HA are formed. The third class includes endo-b-N-acetylhexosaminidases (EC 4.2.2.1), present only in prokaryotes. Bacterial enzymes break b-1,4-glycosidic bonds in HA using a b-elimination reaction. As a result of their action, unsaturated di-, tetra-, and hexasaccharide are formed. There are five types of Hyals found in humans: HYAL-1, HYAL-2, HYAL-3, HYAL-4, and HYAL-5. HYAL-1 and HYAL-2 are found in most tissues and are involved in the circulation of HA 23 . HYAL-2 degrades long-chain hyaluronic acid (HMWHA) found in the extracellular matrix to short-chain hyaluronic acid (LMWHA), which after binding to the CD44 receptor, is transported into the cell. HYAL-1 is found inside the cell and is responsible for the degradation of LMWHA into tetra and hexasaccharide 24 . Hyals are involved in the regulation of important physiological processes, such as fertilisation and skin ageing. Present in sperm, HYAL-5 plays a key role in fertilisation in mammals as it enables the sperm to connect to the egg cell by breaking down HA in the granule layer. The overactivity of collagenases, Hyal, and elastase leads to the formation of wrinkles 25 .
Hyals are an important virulence factor for many bacterial species, such as Staphylococcus spp., Streptococcus spp., and Streptomyces spp.  of body fluids and reduces tissues' integrity, facilitating the penetration of microorganisms and toxins into the skin 26 .
Hyals are an important component of the venom of Hymenoptera, spiders, and snakes. Hyal, present in the venom, helps to distribute the toxins throughout the body. Several studies have shown Hyal to be involved in tumour growth, metastasis, and angiogenesis 27 . However, the conducted studies do not provide unambiguous results showing that Hyals can function both as a suppressor and a promoter of carcinogenesis. HA promotes tumour metastasis, so the enzyme that breaks down hyaluronan (Hyal) inhibits their growth. On the other hand, low molecular weight HA stimulates angiogenesis, promoting tumour growth and metastasis formation 28 .

Polyphenols as inhibitors of hyaluronidase:
structure-activity relationships (SARS)

Flavonoids
Plant-based metabolites represent the chemically-different groups of compounds, of which many have been isolated for the first time from the plants used in the ethnomedicine of indigenous tribes. Those steps have allowed for their identification, biotechnological modification of their structure or to develop a synthesis path involving bacteria or fungi.
Phenolic compounds include a very numerous and important group of compounds commonly found in the world of plants. Polyphenols are plant secondary metabolites with very diverse chemical structures, containing at least two hydroxyl groups attached to an aromatic ring. Due to the number and way of connecting aromatic rings, we can distinguish phenolic acids, flavonoids, stilbenes, and lignans 29,30 . The presence of multiple hydroxyl groups gives phenolic compounds antioxidant properties 31 . Phenolic compounds also show antitumor, anti-inflammatory, antiviral, antibacterial, antifungal, hepatoprotective, antiallergic, anticoagulant, and blood vessel sealing properties 32 . Besides, many plant-derived polyphenols affect Hyal and other enzymes that regulate the metabolism of the extracellular matrix [33][34][35][36] .
Inhibition of Hyal activity by polyphenolic compounds is related to, among others, the presence of hydroxyl groups ( Figure 2). Hertel et al. investigated the effect of flavonoids with a different number of hydroxyl groups on Hyals' activity [flavones (apigenin and luteolin) and flavonols (kaempferol and quercetin) in concentration 0.1 mM]. In the case of bovine testicular Hyal (4 U/mL), the activity of the tested compounds was low (less than 20% for apigenin and luteolin). The inhibition of Hyal was only slightly enhanced by hydroxyl groups (quercetin or myricetin). Phenolic compounds with an additional hydroxyl group in the 3-position (quercetin ca. 90% inhibition or myricetinca. 70% inhibition) more strongly inhibited Streptococcus agalactiae hyaluronan lyase. Additionally, the introduction of a sugar moiety significantly reduces the test compounds' activity (e.g. rutin). Aglycones were more potent inhibitors than their corresponding glycosides, what may probably result from the inhibitor's difficult access to the Hyal active site in case of glycosides 37 .
Another study 38,39 determined the effect of luteolin, apigenin, kaempferol, myricetin, quercetin, and morine on bovine testicular hyaluronidase (BTH). Flavonoids containing a double bond between the 2 and 3 carbon atoms showed a greater potency than flavonoids without the double bond. Additionally, a ketone group in the 4-position or hydroxyl group at 5,7, and 4 0 positions may increase the inhibition of Hyal. In turn, methoxylation of the 4 0 -OH group in hesperitin and diosmetin reduces their activity. On the other hand, the presence of the 3-OH group did not affect the potency of Hyal inhibition. In addition, the presence of a catechol system in the B ring Compounds containing a sugar residue in the C-3 position weakly block Hyal [kaempferol-3-O-a-L-rhamno pyranoside (18.26%), quercetin-3-O-a-L-rhamno pyranoside (6.88%), and quercetin-3-O-a-L-arabino pyranoside (13.81%)] ( Table 2) 42 .
Another possible mechanism involved in an inhibition of Hyals' activity is based on the ability to associate compounds of low molecular weight. The acid function (hydroxyl, carboxyl, phosphate, or sulphate) is necessary to form an aggregate with multiple negative charges. More research is needed to determine the possibility of aggregating by low molecular weight flavonoids that can act as effective inhibitory units.  Bralley et al. determined antioxidants' influence (phenolic acids, flavonoids, and condensed tannins) in Sorghum on Hyal activity. Amongst tested compounds, i.e. condensed tannin, apigenin, luteolin, kaempferol, quercetin, and rutin, only the four first were effective. Probably condensed tannins not only denature the enzyme but also interact with the hydrophobic channel 43 . Tatemoto et al. investigated the effect of tannin, apigenin, and quercetin on Hyal activity and the fertilisation process in vitro. Tannins showed the greatest inhibitory properties at concentrations of 2-10 mg/mL. These data suggest that an adequate concentration of tannic acid prevents polyspermia by inhibiting sperm Hyal activity during IVF of porcine oocytes. However, the presence of apigenin or quercetin in the same concentrations as tannic acid could not prevent polyspermy 44 .
The study by Zeng et al. determined how apigenin, luteolin, kempferol, quercetin, morin, naringenin, daidzein, and genistein bind to HAase. It was shown that those compounds interacted with the active centre of the enzyme through electrostatic forces, hydrophobic interactions, and hydrogen bonds. The binding of flavonoids caused changes in the active centre structure, resulting in inhibition of HAase (Table 3)  Based on the structure of compounds nr 2 and 4, the methoxy groups at the C-3 00 and C-4 00 positions may participate in the Hyal inhibition 46 .
Oligomers composed of caffeic acid exhibited interesting anti-Hyal properties. Caffeic acid trimer isolated from Dracocephalum foetidum inhibited Hyal more strongly than disodium cromoglycate (IC 50 ¼ 220 mM and IC 50 ¼ 650 mM). Similar results were obtained by Aoshima's team studying coffee acid oligomers isolated from Clinopodium gracile. It was appeared that coffee acid oligomers, such as clinopodic acid M, showed more potent inhibitory activity than rosmarinic acid (IC 50 50 values for compounds without the danshensu structure were equal 206 and 653 mM for clinopodic acid J and 8-epiblechnic acid, respectively. An interesting structure for further studies may be clinopodic acid E, which has instead of the structure of 2,3dihydrobenzofuran, 1,4-benzodioxane. This compound is four times more potent than the analogue having the structure of 2,3dihydrobenzofuran (clinopodic acid N). Additionally, as in other studies, a beneficial effect of the increase in oligomer mass on the inhibition of Hyal activity is seen 40,47 .

Tannins
Tannins are nitrogen-free plant substances of high molecular weight (500 À 3000), having numerous hydroxyl groups. Due to their chemical structure, they are divided into two groups: hydrolysing and non-hydrolysing (condensed) ( Figure 5). The first group is divided into galotannins (ester combinations of gallic acid and its derivatives) or elagotannins (ester combinations of ellagic acid). The second group is formed by the condensation of catechins (flavan-3-ol products). Tannins show the ability to form complexes with proteins, resulting in an astringent effect on the skin and mucous membranes. Tannins have been shown to have many properties, such as antibacterial, antiviral, anticancer, antioxidant, anti-inflammatory, and anti-hemorrhagic [49][50][51] .
In another study 53 , gallic acid esters with different n-alkanol chain lengths (from C-1 to C-12) were examined to determine their inhibitory activity against Hyal. With an increase of the alkyl chain length, the inhibitory activity was increased. Cromoglycan disodium (IC 50 ¼ 450 mM) was used as a positive control. Hexyl (IC 50 ¼ 253 mM), heptyl (IC 50 ¼ 112 mM), octyl (IC 50 ¼ 106 mM), nonyl (IC 50 ¼ 167 mM), and decyl (IC 50 ¼ 580 mM) gallates inhibited Hyal. Next, the impact of hydroxyl groups in octyl gallate on an activity was checked. Octyl 3-hydroxybenzoate and octyl 4-hydroxybenzoate did not inhibit Hyal. Octyl 3,4-dihydroxybenzoate (IC 50 ¼ 902 mM) blocked the enzyme to a small extent. The strongest  inhibitor was octyl 3,5-dihydroxybenzoate (IC 50 ¼ 113 mM). This shows the significance of the 3,5-OH grouping in gallic acid ( Table  5). The type of inhibition was determined only for octyl gallate, which inhibited the enzyme in a competitive manner. The K i value was estimated to be 45 mM ( Figure 6). Tokeshi et al. examined the effect of three tannins (tannic acid TA, gallic acid GA, and ellagic acid EA) on boar sperm Hyal. TA and EA strongly inhibited Hyal in the concentration range of 2-10 mM 54 . The phlorotannins present in Eisenia bicyclis and Ecklonia kurome inhibited Hyal activity more strongly than standard substances such as disodium cromoglycate (IC 50 ¼ 270 mM), catechin (IC 50 ¼ 620 mM), and epigallocatechin gallate (IC 50 ¼ 190 mM). The IC 50 values for phloroglucinol, phloroglucinol tetramer, eckol (trimer), phlorofucofuroeckol A (pentamer), dieckol, and 8,8 0 -bieckol (hexamers) were at the level of 280, 650, >800, 140, 120, and 40 mM, respectively. In the case of phlorofucofuroeckol A, dieckol, and 8,8 0 -bieckol an inhibition type (competitive inhibition) and the inhibition constant (K i ) values (130,115, and 35 mM, respectively) were also established. Additionally, it was confirmed that the higher molecular weight of inhibitor the stronger inhibition is observed. This is probably associated with a stronger effect on the dimensional structure of the enzyme 55 .

Alkaloids
Natural alkaline nitrogen compounds are synthesised by plants, fungi, bacteria, and animals. Alkaloids, in minimal doses, have a strong physiological effect, especially on the central nervous system. Moreover, these compounds have antitumor, anaesthetic, antifungal, antibacterial, anti-inflammatory, and analgesic properties. Due to the heterocyclic ring system, we distinguish derivatives: pyridine and piperidine, tropane, quinoline, quinoline, indole, ergot, and purine 56 . A study by Girish et al. determined the effect of aristolochic acid on the activity of purified Indian cobra venom hyaluronidase (NNH1) and the activity of whole venom Hyal. The tested compound inhibited NNH1 non-competitively. Besides, the venom's administration with aristolochic acid to mice showed more than a twofold increase in survival time compared to mice injected with the venom alone. Lower survival was obtained by splitting the application of the inhibitor over time (10 min). Aristolochic acid did not bind to the enzyme's  active site but interacted with exposed tyrosine and tryptophan HAase residues. Aristolochic acid (50, 100, and 200 mM) inhibited NNH1 at the level of 100% for each concentration. Other alkaloids, ajmaline, and reserpine inhibited Hyal weaker than aristolochic acid (ajmaline 11, 26, 40%; reserpine 9, 23, 31%, respectively) ( Table 3) 57 .

L-ascorbic acid
Ascorbic acid (vitamin C -AA) is a compound commonly found in the world of plants and animals. Human is incapable of synthesising vitamin C, therefore, it must be supplied in the diet (parsley, red pepper, black currant, and Brussels sprouts). The ability of vitamin C to create an oxidative system (AA < ¼> AA < ¼> dehydroascorbic acid) determines its antioxidant properties. AA is the most important antioxidant of extracellular fluids in the human body. It is present in high concentrations in the eyeball and lymphocytes, protecting cells against reactive forms of oxygen and nitrogen. Besides its antioxidant activity, AA is involved in the absorption of non-heme iron; in the metabolism of fats, cholesterol, and bile; regeneration of vitamin E in the cell membrane; in the synthesis of collagen, accelerating the wound healing process. Additionally, the presence of AA in the skin may constitute a defense mechanism against an invasion of pathogenic bacteria. Despite the fact that AA is one of the most known biologically-active compound its anti-Hyal activity and SAR are still unknown in details. Several studies involving AA and AA derivatives have found to be able to inhibit Hyals [59][60][61] .
In 2001, Li et al. first described a competitive type of AA inhibition on Hyal isolated from Streptococcus pneumoniae LHyal (hyaluronan lyase). The activity of AA towards Hyal is due to the structural similarity of vitamin C to glucuronic acid, being one of the basic building blocks of hyaluronan (HA) (b-1,4-glucuron-b-1,3glucosamine). It was found that one AA molecule may bind to the enzyme's active site. The AA carboxyl group provides a negative charge that directs the molecule to the positively charged enzyme gap. In the active centre of the enzyme, AA interacts with amino acids through hydrophobic (Trp-292), ionic (Arg-243, Arg-462, and Arg-466), and hydrogen (Tyr-408, Asn-290, and Asn-580) bonds 62 .
In 2003, Okorukwu et al. confirmed the inhibitory effect of AA and AA analogs on the activity of bovine testicular Hyal (BTHfinal concentration 3.5 Units/mL) and LHyal (Streptococcus zooepidemicusfinal concentration 2.5 Units/mL). Gel permeation chromatography (GPC) was used in this study to evaluate the inhibitory activity. The AA and AA derivatives (D-iso-ascorbic acid and dehydroascorbic acid) blocked the hyaluronan lyase more strongly than BTH. D-Saccharic-1,4-lactone and saccharic acid inhibited LHyal without affecting the enzymatic activity of testicular Hyal. The introduction of a carboxyl group that gives the molecule a negative charge positively affects the inhibitory effect of AA derivatives. Hydrogenation of the double bond between the 2nd and 3rd carbon atoms decreases the activity of the compounds. Saccharic acid can be used to develop selective inhibitors of bacterial hyaluronan lyase (Table 8) 63 .
In a study conducted by Botzki et al., a positive correlation was confirmed between the inhibition of Hyal activity and the increased hydrophobic interactions. L-Ascorbyl palmitate, through an increase in hydrophobic interactions with Phe343, His399, and Thr400 in the active centre, led to increased inhibition of hyaluronan lyase (competitive inhibition). A similar effect was achieved with BTH. The long alkyl chain interacts with a hydrophobic channel formed primarily by the amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 64 .
The new LHyal inhibitors should have a larger ring system to favourably influence the hydrophobic bonding to the Trp-292 indole group and contain at least one negative charge group (carboxyl group), which brings the inhibitor to the cleft region (rich in positively charged arginine).
Spickenreither et al. examined the effect of 6-O-acylated AA derivatives on the Hyal activity of BTH and Streptococcus agalactiae strain 4755 (Sag Hyal 4755). All compounds showed more potent activity against bacterial lyase. Methylation of the endiol system reduces the activity of vitamin C analogs. On the other hand, 2 and 3 dibenzylated derivatives showed more potent inhibitory properties than AA. The increase in potency is due to additional hydrophobic interactions between the rings and the active centre. An increase in the length of the 6-O-acyl residue (13 b-j) results in increased inhibitory activity. The IC 50 for octadecanoate was 0.9 and 39 mM for BTH and Sag Hyal 4755, respectively. Shortening of the aliphatic chain and adding phenyl, p-phenylene, or p-biphenyl groups leads to compounds with comparable inhibitory properties. Additionally, ether bonds in the synthesis of new inhibitors positively influence their activity. This             is associated with the formation of additional hydrogen bonds in the active centre ( Figure 8; Table 9) 65 .

Glycosides
Glycosides are a group of the organic compounds consisting of sugar and an aglycone part. The bond between the sugar and the aglycone is called a glycosidic bond. The chemical nature of aglycones is very different, they can be alcohols, lactones, phenolic acids, thiols, etc. The sugar portion may consist of 1-12 monosaccharide, disaccharide, or oligosaccharide molecules. The glycosides widely occur in the plant world, especially in higher plants.
Because of the chemical diversity of glycoside, the plant-based sources of glycosides are important in phytotherapy 66 (Table 10). The samples were divided into 5 equal parts and mixed with linamarine at doses of 0.5, 0.75, 1, and 2 mM and with amygdalin at doses of 0.4, 0.8, 1, and 2 mM. Incubation of compounds with sperm resulted in a significant reduction (dosedependent) of sperm motility and Hyal activity compared to the control group (isotonic saline solution). Both linamarine and amygdalin did not change sperm morphology. The authors concluded that the fertilisation ability of bull sperm could be inhibited by the over-consumption of plants rich in cyanogenic glycosides 67 .

Terpene/terpenoids
Terpenes are composed of a varying number of isoprene units ( Figure 9). They are commonly found in the plant world in hydrocarbons or oxidised forms (with hydroxyl, carbonyl, or carboxyl groups). Depending on the number of isoprene residues, we distinguish monoterpenes (C10), diterpenes (C-20), sesquiterpenes (C-15), triterpenes (C-20), and tetraterpenes (C-40). Due to their highly diverse chemical structure, terpenes exhibit a variety of biological activities, such as antibacterial, antiviral, anticancer, antiinflammatory, and sedative effects 68  Depending on the type of sapogenin we distinguish triterpene saponins, steroidal saponins, and steroidal alkaloids ( Figure 9). These compounds reduce the surface tension of water solutions.     They show anti-inflammatory, antibacterial, protozoal, antifungal, and antiviral activity, stimulate secretion of gastric juice, bile, and intestinal juice. They can affect cholesterol level 70 . In a study by Zhou et al., they determined the effects of esculeoside A and its aglycone esculeogenin A on Hyal activity in vitro and in mice dermatitis model. Esculeoside A, a spirosolate-type glycoside, is identified as a significant component of ripe tomato fruit. The IC 50 for esculeogenin A and esculeoside A was about 2 and 9 mM, respectively. Administration of esculeoside A at a dose of 10 mg/kg for four weeks to mice with dermatitis significantly reduced diseases symptoms 71 . Esculeoside A is a competitive inhibitor of hyalurnidase (K i ¼ 11.0 mM) 72 . Also, the other steroid alkaloids present in tomato juice showed beneficial inhibitory effects against Hyal. Administration of 10 mg/kg esculeoside B to mice with dermatitis for four weeks significantly reduced skin inflammation. In addition, it was found that esculeoside B administration significantly inhibited T-lymphocyte proliferation and decreased IL-4 production 73 .
More information about the effect of saponin structure on Hyal activity was provided by QSAR studies of ursolic and oleanolic acids, the results of which exhibited the higher activity of ursolic acid than oleanolic acid. In that experiment, the effect of the position of methyl groups at 29 and 30 carbon atoms was checked. Both geminal and vicinal positions had no significant impact on an inhibitor activity. For oleanolic acid, the activity increased when the methyl group was introduced at C-17 or C-16 and decreased when the methoxyl group was introduced at C-23. The 3-OH acetylation reduced the activity of the compounds. Carboxylation of C-30 increased the activity of the compounds, while esterification of the same carbon (C-30). In addition, the introduction of a sugar moiety into the 3-OH decreased their activity. In the case of ursolic acid, the activity decreased when the hydroxyl group was modified at C-3 (3-oxo, 3-hydroxyimino, and 3-acetylate derivatives) and at C-28. Replacement of the methyl group at C-23 with a carboxyl or hydroxy methylene group decreased the activity of the compounds. As in the case of oleanolic acid, the introduction of a sugar group caused a decrease in the inhibitory activity ( Figure 10; Table 11) 74 .
The absence of an OH group at the C-3' or C-4' position reduces inhibitory activity The introduction of a sugar residue reduces inhibitory activity dramatically Removal of the 3-OH groups has no significant effect on the inhibitory activity Methylation of the OH group at C-3' or C-4' increases inhibitory activity Figure 5. Potential groups engaged in an interaction fukiic acid derivative-hyaluronidase.    Similar results were obtained in another experiment aimed at an investigation of the impact of the oleanic acid structure modification on anti-Hyal activity. Oxidation of the 3-OH group in oleanic acid led to a decrease of the activity about 4-fold (59.3-15.9%). A similar effect was obtained by esterification of the COOH group (12.7% inhibition). The activity of the compounds was evaluated at a concentration of 40 mg/mL 75 .

OH
In another study 76 , oleanane-type saponins, isoflavonoids, oxazoles, and glycosides (36 compounds) were isolated from the aerial part of Oxytropis lanata (Pall.) DC. The effect of saponins against Hyal (IC 50 ¼ 0.15-0.22 mM) was more potent than sodium cromoglycate, which was used as a positive control (IC 50 ¼ 0.37 mM). On the basis of the structure analysis, it was appeared that all isolated saponins contain a 3-O-b-D-glucuronopyranoside grouping in the sugar moiety, which shows a potent inhibition of Hyal, as well as the compounds with a ketone group at C-22 more strongly blocked the enzyme ( Figure 11). Similar results were obtained when testing triterpene saponosides from the 3-O-b-Dglucuronopyranoside group, which blocked Hyal more strongly (2; IC 50 ¼ 1.25 mM, 3: IC 50 ¼ 0.68 mM, and 9: IC 50 ¼ 0.82 mM) than rosmarinic acid used as a control (IC 50 ¼ 1.36 mM) 77 . The significance of the 3-O-b-D-glucuronopyranoside grouping was also noted by examining the triterpene saponosides camelliagenin A (IPS-1 and IPS-2) isolated from the methanolic root extract of Impatiens parviflora DC. A very interesting result has been obtained, because IPS-2 (IC 50 ¼ 286.7 mg/mL) inhibited BTH Hyal activity more strongly than escin (IC 50 ¼ 303.93 mg/mL). What is interesting, escin is recommended to be used as an anti-Hyal reference compound. The higher IC 50 value was obtained for IPS-1; 368.1 mg/mL. Considering the structure of these compounds, a clear difference may be noticed significantly since the tested saponosides differed in their acetylation at C-16. The 16-O-acetylcameliagenin A derivative showed less activity. Therefore, the free OH group at position 16 may have a beneficial effect on Hyal inhibition, e.g. via participation in ions chelating in the reaction's medium 78 . Another study examined how sugar moiety in saponins affects the activity of Hyals isolated from different bacterial species (Streptococcus agalactiae -Hyal B, Streptomyces hyalurolyticus -Hyal S, Streptococcus equisimilis -Hyal C) and bovine testes (BTH). Glycyrrhizin (1) and its aglycone glycyrrhetinic acid (2) were used as inhibitors. The tested compounds most potently blocked the activity of Hyal B [(1); IC 50 ¼ 0.440 mM, (2); IC 50 ¼ 0.060 mM]. For other Hyals, the action was weak [Hyal S -(1); 1.020 mM, (2); 0.260 mM; Hyal C -(1); NA, (2); NA; BTH -(1); 1.300 mM, (2); 0.090 mM). Considering the results obtained, glycyrrhetinic acid inhibited the activity of tested enzymes weaker, which may prove the significance of the sugar moiety in the inhibition of Hyal activity 25   Increasing the length of the alkyl chain increases the activity of the inhibitors The introduction of methyl groups reduces the activity of inhibitors The olean-type saponins, spinasaponin A (lack of activity), spinasaponin A 28-O-glucoside (IC 50 ¼ 620 mM), udosaponin B (IC 50 ¼ 750 mM), and sandrosaponin IX (IC 50 ¼ 370 mM), isolated from the roots of Oenanthe javanica, showed moderate ability to inhibit Hyal. Esterification of the 28-COOH group with b-D-glucopranosyl increased the activity of the compounds 81 .

Tyrosine and tyrosinase
Tyrosinase is a key enzyme involved in melanogenesis. This enzyme belongs to the class of oxidoreductases (EC 1.14.18.1). It is responsible for the catalysis of tyrosine hydroxylation to L-DOPA and the oxidative conversion of L-DOPA to dopaquinone ( Figure  12). The active centre of tyrosinase consists of two copper atoms linked by a coordination bond with three histidine residues. Three different types of tyrosinase are involved in melanin production: oxy-tyrosinase, met-tyrosinase, and deoxy-tyrosinase. Oxy-tyrosinase and met-tyrosinase have Cu (II) copper atoms in their active centre, and deoxy-tyrosinase has two Cu (I) atoms. Deoxy-tyrosinase does not perform a catalytic function, but it is easily converted into the oxy form, which is the only form of the enzyme capable of transforming both monophenol and diphenol substrates. On the other hand, met-tyrosinase is formed during the reaction catalysed by the oxy form and is responsible only for reactions with diphenolic substrates (Figure 13) [81][82][83][84] .
Tyrosinase is an enzyme widely distributed in nature, including in fungi and to a lesser extent in bacteria and algae. This enzyme is also found in plants and animals. In higher vertebrates, melatonin is produced in melanocytes present in the epidermis, hair follicles, the uveal membrane of the eye (choroid, ciliary body, and iris), the inner ear (cochlea), and the central nervous system (the arachnoid and internal filum terminale). Melanins are macromolecular polymeric pigments resulting from the oxidation and polymerisation of phenolic compounds ( Figure 14). Melanin synthesis takes place in vesicles called melanosomes and it is considered to be one of the most common pigments in nature. In mammals, melanosomal tyrosinase is involved in the formation of black-brown eumelanin and yellow-reddish phaeomelanin. Eumelanin has photoprotective properties, resulting from the ability to absorb ultraviolet radiation (UV) and neutralise free radicals and reactive oxygen species (ROS). On the other hand, pheomelanin has photosensitising properties and, under the influence of UV radiation, can participate in the generation of ROS. In mammals, melanin pigmentation performs many critical physiological tasks, such as adaptive colouration, protection of essential tissues against UV radiation, thermal control of the organism, regulation of vitamin D 3 biosynthesis. Abnormal tyrosinase activity is responsible for skin abnormalities such as vitiligo or freckles. Also, tyrosinase may play a role in carcinogenesis and neurodegenerative diseases such as Parkinson's disease. Tyrosinase also contributes to the formation of brown colour in fruits and vegetables due to the reaction of dopaquinone with amino acids and proteins present in these foods. In most studies on the inhibition of tyrosinase activity, fungal tyrosinase was used due to its widespread availability. The enzyme isolated from the mushroom A. bisporus is very similar in structure to tyrosinase occurring in mammals, making it a suitable model for studying the process of melanogenesis. Since tyrosinase is a reasonably significant target in agriculture, food, medicine, and cosmetology, much attention has been paid to the development and screening of tyrosinase inhibitors [85][86][87] . Table 8. Structures of L-ascorbic acid and its derivatives with an anti-hyaluronidase activity.

Polyphenols as inhibitors of tyrosinase: a structure-activity relationship
The positive effect of polyphenols on human health is mainly related to their antioxidant properties. The antioxidant activity of individual polyphenols depends on the number of hydroxyl groups and their location. It has been shown that the more hydroxyl groups in a molecule, the more potent antioxidant activity. Compounds with redox properties effectively prevent melanin biosynthesis due to their multidirectional mechanism of action. Tyrosinase inhibition by polyphenols is based on free radical scavenging properties and the ability to chelate copper in the tyrosinase active site.

Phenolic acids
Phenolic acids are one of the most common groups of organic compounds present in plants. They are made of a phenolic ring and a carboxylic acid residue. There are two subclasses of phenolic acids: derivatives of benzoic acid and cinnamic acid. Several studies have shown their inhibitory effect on tyrosinase activity and that activity was related to the number and position of hydroxyl groups 88 . Considering the structure of phenolic acids, three mechanisms of tyrosinase inhibition can be distinguished. The first is related to the chelation of copper ions in the active centre. The second mechanism is associated with the disturbance of the enzyme's tertiary structure through hydrogen bond formation. The third mechanism involves constructing hydrogen bonds between the hydroxyl groups of phenolic acids and the carbonyl oxygen of the Tyr98 ORF378 protein, preventing the interaction between tyrosinase and ORF378. Consequently, ORF378 cannot serve as a Cu (II) ion transporter to the enzyme's active site 89,90 .

Hydroxybenzoic acids.
Kubo et al. investigated the effect of anisic acid and its derivatives on L-DOPA oxidation by tyrosinase. As the concentration of anisic acid increased, the enzymatic activity decreased sharply but was not completely inhibited (IC 50 ¼ 0.60 mM). The inhibition of fungal tyrosinase by anisic acid is a reversible reaction in which the tested acid is a non-competitive inhibitor (K i ¼0.603 mM). The modification of the alkyl chain in anisic acid influenced the activity and type of inhibition of the tested compounds. P-ethoxybenzoic acid showed a kind of incompetent inhibition, p-propoxybenzoic acid was of mixed, and pbutoxybenzoic acid was of competitive. With the increase in the alkyl chain's length, the inhibitory activity of the tested compounds decreased 91,92 . Chen et al. obtained similar results when testing p-alkoxybenzoic acid derivatives. The tested compounds behave as reversible tyrosinase inhibitors in the presence of L-DOPA substrate (k 475 nm; spectrophotometric method, 6680 U/mg). Among them, p-hydroxybenzoic acid (IC 50 is of the mixed type, and the others show a type of non-competitive inhibition (p-propoxybenzoic acid, pbutoxybenzoic acid, p-pentoxybenzoic acid, and p-hexyloxybenzoic acid). Additionally, it was appeared that increasing the chain length above two carbon atoms changed the type of braking from competitive to non-competitive 93 . (22) Another study 94 determined the effect of methoxylation of hydroxyl groups on the tested acids' activity and their esters. Protocatechuic acid methyl ester, protocatechuic acid, vanillic acid methyl ester, vanillic acid, isovanillic acid methyl ester, isovanillic acid, veratric acid methyl ester, and veratric acid were used in the study. Only protocatechuic acid and its methyl ester inhibited the enzyme (60.1 and 75.4% inhibition; ID 50 ¼0.42 mmol/mL and 0.28 mmol/mL). The hydroxyl group at the para and meta position is an important part of the structure of inhibitors. Protocatechuic acid methyl ester inhibited the enzyme activity more strongly than protocatechuic acid, which may result from the esterification of the carboxyl group. Kubo et al. provided more information about the impact of an esterification on the activity of gallic acid. It was appeared that, apart from gallic acid (4.5 mM), the IC 50 of all esters was almost comparable (<0.5 mM). Based on the above observation, it can be concluded that gallic acid esters with an increase in the number of carbon atoms of the alkyl chain (>C10) may be more challenging to incorporate into the protein pocket to reduce the rate of oxidation by the enzyme. Hence, gallates with a longer alkyl chain (>C-10) become inhibitors but not substrates, which indicates that gallates with a longer alkyl group (>C-10) can be expected as more suitable inhibitors. However, tetradecanoyl gallate (C-14) and hexadecanyl gallate (C-16) are sparingly soluble in water ( Figure 12; Tables 1S and Table 13   Another study 97 assessed the influence of methoxylation of cinnamic acid and its derivatives (cinnamic acid, 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, 3,4-dihydroxycinnamic acid, 2-methoxycinnamic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic acid; concentrations from 0.1 to 3 mmol/L) on the action of tyrosinase (3130 U/mg) in the presence of L-tyrosine or L-DOPA as a substrate. Cinnamic acid (IC 50 ¼2.1 mM), 2-hydroxycinnamic acid (IC 50 ¼0.5 mM), and the Omethyl (IC 50 ¼0.42 mM) forms exhibited inhibitory properties compared to 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, and 3,4-dihydroxycinnamic acid, which turned out to be substrates of tyrosinase. All inhibitors showed competitive inhibition. The inhibition constants are about the same for the oxidation of L-tyrosine and L-DOPA, indicating that the inhibitors bind to the same form of the enzyme. In the next study 98 , the inhibitory effect of caffeic acid and ferulic acid on tyrosinase activity isolated from murine B16 melanoma cells (90 U) was analysed. Both ferulic acid (27.4% non-toxic conc.) and caffeic acid (24.4% non-toxic conc.) effectively inhibited melanin production in B16 melanoma cells. Ferulic acid was reducing tyrosinase activity by binding directly to the enzyme, whereas no binding was observed between caffeic acid and tyrosinase.
One of the important structural elements regulating the enzyme's activity is a type of bonds present in the inhibitor's structure. Some compounds, such as p-coumaric acid (IC 50 ¼115.6 mM; % inhibition 74.4) and isoferulic acid (IC 50 ¼114.9 mM; % inhibition 77.8) lose their tyrosinase inhibitory properties after saturation of the double bond (dihydro-p-coumaric acid IC 50 ¼1000 mM; 74.4% inhibition and dihydroisoferulic acid IC 50 ¼195.7 mM; 60.6% inhibition). None of the compounds tested were more effective than kojic acid (IC 50 ¼51.6 mM) or arbutin (IC 50 ¼210.5 mM). All compounds tested show a noncompetitive type of inhibition. The results indicate that the reduction of double bonds weakens the inhibitory activity of the test compounds. The C ¼ C binding is necessary for the proper interaction of the inhibitor with the active site 99 .
In another study, the type of inhibition and the effect of caffeic acid, p-coumaric acid, and rosmarinic acid on monophenolase and diphenalase activities were determined. The most active compound was p-coumaric acid (L-tyr, IC 50 Tables 2S  and Table 13) 100 .  The addition of a hydroxyl, methyl or carbonyl group reduces the activity of the inhibitors Removal or modification of the hydroxyl group will reduce the activity of the inhibitors Removal or modification of the hydroxyl group will reduce the activity of the inhibitors Esterification of the hydroxyl group reduces the activity of the inhibitors; carboxylation increases the activity of the compounds Figure 10. Chemical groups of triterpenic acids involved in the inhibition of hyaluronidase. Flavonols, due to the additional hydroxyl group at the C-3 position, block tyrosinase much more strongly than flavones 102 . Kim et al. provided more information regarding the influence of the position and number of hydroxyl groups on their inhibitory activity. Natural and synthetic flavones and flavonols were used in the study. L-tyrosine was used as a substrate for fungal tyrosinase. It appears that the hydroxyl groups at the C-7 (ring A) and C-4 0 (ring B) positions may increase the inhibitory activity of flavonoids. In this study, 3 0 ,4 0 ,7,8-tetrahydroxy flavone showed the strongest inhibitory properties (IC 50 ¼0.07 mM). Two catechol groups in ring A and B are probably responsible for the inhibition. Lack of the fourth hydroxyl group in 4 00 ,7,8-trihydroxy flavone (IC 50 ¼12.95 mM) and 3 00 ,4 00 ,7-trihydroxy flavone (IC 50 ¼24.1 mM) weakens their activity. The 5-OH group also has a significant influence on the activity of the inhibitors, e.g. 2 0 ,5,7-trihydroxyflavone (IC 50 ¼12.95 mM) and 3 0 ,4 0 ,5,7-tetrahydroxy flavone (IC 50 ¼12.95 mM) block tyrosinase more strongly than 2 0 ,7-dihydroxy flavone, and 3 0 ,4 0 ,7-trihydroxy flavone. Also, the hydroxyl group in the C-3 position, which divides flavonoids into flavones and flavonols, influences on the activity with a weaker inhibition for flavones. Additionally, an increase in the number of hydroxyl groups in flavonoids reduces their inhibitory activity against tyrosinase (3 0 ,4 0 ,5,6,7-pentahydroxy flavonol IC 50 ¼314.23 mM). The location of the hydroxyl groups plays a more important role than their number. Summarising the increase in the number of hydroxyl groups on the other side of the flavonoids (C-7, C-8, C-2 0 , C-3 0 , and C-4 0 ) increases compounds' activity 103 . In contrast, an increase in the number of hydroxyl groups on the ketone side (C-5, C-6, C-5 0 , C-6 0 ) reduces the activity of inhibitors, which is related to the disturbance of the interaction with tyrosinase. Isoflavonoids are characterised by a linked ring B at the third carbon atom. The location and number of hydroxyl groups in the A-ring of isoflavonoids can strongly affect both the inhibitory power and inhibition type. 6,7,4 0 -trihydroxyisoflavone, daidzein, glycitin, daidzin and genistin showed strong monophenolase inhibitory activity but weak diphenolase inhibitory activity. 4 0 ,6,7-trihydroxyisoflavone (IC 50 ¼9 mM) shows the strongest properties. Presence of a single hydroxyl group in the C-7 position (4 0 ,7-trihydroxyisoflavone IC 50 ¼203 mM; 6-methoxy-7,4 0 -dihydroxyisoflavone IC 50 ¼218 mM) or no hydroxyl groups (4 0 -hydroxyisoflavone-7-O-glucoside IC 50 ¼267 mM) in ring A significantly reduces the activity of the compounds. The presence of the hydroxyl groups in the C-7 and C-8 position can influence the type of inhibition, shifting from reversible to irreversible type 104 . Similar results were obtained in case of anthocyanins and an assessment of the impact of the number and distribution of hydroxyl groups in the B ring on tyrosinase activity. The following compounds were investigated: pelargonidin (IC 50 ¼66 mM), cyanidin (IC 50 ¼27.1 mM), and delphinidin (IC 50 ¼57.4 mM) in the presence of kojic acid (IC 50 ¼34.8 mM) as a positive control. The substrate for the reaction was L-DOPA. These results indicate that the structure with two hydroxyl groups in ring B has the greatest inhibitory effect 105 .

Flavonoids
Another study examined the effect of isoflavonoids isolated from the roots of Pueraria lobata, such as daidzein and formononetin. These compounds have appeared to be of weak inhibitors with the IC 50 values for daidzein L-tyr. 350 mM; L-DOPA 350 mM and for formononetin L-tyr. IC 50 >350 mM; L-DOPA IC 50 >350 mM. A significant increase in an activity was obtained by introducing OH groups into the B ring at the C-3 0 position and methylation of the 4 0 -OH group -calycosin (L-tyr. IC 50 ¼7.02 mM; L-DOPA IC 50 ¼1.45 mM). This compound is more active than kojic acid (Ltyr. IC 50 ¼12.10 mM; L-DOPA IC 50 ¼9.14 mM). It is seen that the presence of a hydroxyl group at the C-3 0 position and a methoxyl group at the C-4 0 position of the isoflavone backbone plays a major role in the anti-tyrosine activity 106 . The inhibitory activity of calycosin against tyrosinase (monophenolase) was also confirmed by Kim et al. with the IC 50 equal of 38.4 mM. In turn, the IC 50 value for kojic acid and arbutin was 51.5 and 120.9 mM, respectively 107 . However, calycosin was more toxic than standards (LD 50 ¼120 mM vs. LD 50 >200 mM for kojic acid and arbutin). The results of another study conducted by Kim et al. have also shown an inhibitory activity of calycosin with the IC 50 ¼30.8 mM and for kojic acid IC 50 ¼50 . 1 mM 108 .
The isoflavonoids isolated from the stem of Maackia fouriei have also exhibited their anti-tyrosinase activity. Methylation of the 4 0 -OH group in formononetin, texasin, and odoratin, resulted in impaired anti-tyrosinase properties. The presence of 5-OH group in genistein (IC 50 ¼33 mM) and tectorigenin (IC 50 ¼20 mM) favourably affects the activity of isoflavonoids in comparison to daidzein (IC 50 ¼41 mM) without 5-OH group. An interesting compound isolated from M. fouriei is the bishomoflavonoid derivative, mircoin (IC 50 ¼5 mM). This compound inhibited the enzyme competently. Due to its strong inhibition, further structure-activity studies are needed for this compound. In this study, kojic acid (IC 50 ¼45 mM) was used as a positive control 109 .
In another study, the effect of OH groups at the C-6, C-7 and C-4 0 positions on isoflavonoid activity was investigated. 6,7,4 0 -Trihydroxyisoflavone inhibited tyrosinase competently (IC 50 ¼9 mM; K i value of 5.72-6.24 mM). Methylation of the 6-OH group in glycitein (IC 50 ¼264 mM), the absence of the 6-OH group in daidzein (IC 50 ¼237 mM), or the presence of an OH group at the 5-OH position in genistein (IC 50 ¼822 mM), decreased the anti-tyrosinase activity (Table 14) 110 .
The discrepancies in the study are due to the variation in experimental conditions such as temperature, pH, and concentrations of enzyme and substrates used. Flavonols are the most potent tyrosinase inhibitors among flavonoids. This is due to the similarity of flavonols to the structure of kojic acid À 3-hydroxy-4keto moiety (Figures 16 and 17).

Double bond.
The double bond between the second and third carbon atoms is preferred for flavonoids to maintain a flat molecular structure. Naringenin (IC 50 ¼c. 555 mM) showed less inhibitory activity than apigenin (IC 50 ¼c. 40 mM). Dihydromyricetin (IC 50 ¼c. 37 mM) showed greater inhibitory activity than myricitin (IC 50 ¼c. 85 mM), while taxifolin (IC 50 ¼c. 800 mM) showed less inhibitory activity than quercetin (IC 50 ¼c. 30 mM). These results suggested that C2 ¼ C3 binding affects the inhibitory properties of the flavonoids 102 .  (Tables 3S and  Table 16) 102 .  Comparing baicalein (IC 50 ¼290 mM) with chrysin (no activity), the other hydroxyl group at the C-6 position of baicalein results in more potent tyrosinase inhibition. Comparing the inhibitory potency of baicalein (aglycone) with its glycosides, oroxin B (no activity) and oroxin A (IC 50 ¼500 mM), a decrease in an inhibitory activity was noted. Glycosylation of the hydroxyl group at C7 was negatively correlated with the inhibitory activity of flavonoids. In addition, the activity of glycosides was influenced by the type of sugar moiety. The presence of the b-D-gentiobiosyl group reduced the inhibitory activity stronger than b-D-glucopyranosyl. This effect is due to spherical collapses 114 .

Lignans
Lignans are phenylpropanoid dimers belonging to the group of plant phytoestrogens ( Figure 18). These compounds are widespread in seeds (lentils), vegetables (garlic and asparagus), and fruits (pears and plums), however, the richest source is linseed and whole cereal grains. They are part of the cell wall and can be released by intestinal bacteria. Due to the similar structure to oestrogens, lignans compete for oestrogen receptors. In oestrogen deficiency, lignans gently complement their action, and when there is an excess of them, they reduce their activity because they have a much weaker oestrogenic effect. As a result, they help maintain the hormonal balance in the body and reduce the risk of various hormone-dependent diseases. Besides, these compounds protect against osteoporosis, lower LDL cholesterol, inhibit bacteria and fungi' growth, and lower blood glucose levels. The most important compounds in this group are sesamine, sesaminol, sesamoline, pinoresinol, secoisolaricresinol, matairesinol, schizandrin, and schizandrol [115][116][117][118][119][120] .
Eight lignans were isolated from the methanolic extract of    Lignan glycosides showed a moderate inhibitory effect on tyrosinase (4-5 times less than kojic acid) in the presence of L-DOPA as a substrate (4-O-lariciresinol-glucoside À 17.74% inhibition and 4 0 -O-lariciresinol-glucoside À 12.27% inhibition). When compared to lignan diglucoside (11.06% inhibition), they showed a lower activity, probably due to the complete absence of free hydroxyl groups (Table 17) 112 .

Flavonolignans
Phytochemicals composed of part flavonoid and part phenylpropanoid, which are commonly found in nature. The richest source of these compounds is Silybum marianum from the Asteraceae family. They show hepatoprotective, anticancer, and anti-inflammatory effects 123 .

Stilbenes
These compounds belong to phytoalexins, low molecular weight cell components with antibacterial and antifungal properties.
Besides, they show other biological properties such as antioxidant, anti-inflammatory, and antiproliferative effects. A characteristic feature of their structure is the presence of a 1,2-diphenylethylene core. More than 400 natural stilbenes have been discovered, but due to the low abundance of the critical enzyme stilbene synthase, they are not widely distributed in nature. The primary source of stilbenes in the human diet are grapes, red wine, and peanuts. The most famous representative of this group is resveratrol 125 .

Hydroxyl groups.
Many naturally occurring stilbenes exhibit tyrosinase inhibitory activity, that is related to the characteristic elements of their structure. The inhibitory properties are due to the number and distribution of oxygen atoms attached to the aromatic rings. Dioxyl stilbene, pinosylvin, showed weak inhibitory properties (IC 50 ¼46 mM), while resveratrol, a stilbene representative with three hydroxyl groups, inhibited tyrosinase even more strongly than kojic acid. However, when compared to oxyresveratrol, representative of tetroxyl stilbenes, that compound showed a nine-fold increase in the inhibition than resveratrol (IC 50 ¼1.5 vs. 14.4 mM) 126 . To better understand, the structure-activity relationship in a model hydroxystilbene-tyrosinase new derivatives of trans-stilbene were synthesised 127 . Monohydroxy trans stilbenes showed no inhibitory effect on tyrosinase, only after attachment of another hydroxyl group to the aromatic ring resulted in an increase of inhibition. The braking force depended on the position of the hydroxyl groups to each other, e.g. 3,3 0dihydroxy-transstilbene (26.3% inhibition IC 50 >200 mM) has a more substantial inhibitory effect than 2,3-dihydroxy-trans-stilbene (4.4% inhibition IC 50 >200 mM) and 3,4-dihydroxy-trans-stilbene (9.5% inhibition IC 50 >200 mM) or 3,5-dihydroxy-trans-stilbene (18.6% inhibition IC 50 >200 mM). The 3,3 0 ,4-trihydroxy-trans-stilbene (87.7% inhibition IC 50 ¼74.3 mM) and 3,3 0 ,4,4 0 -tetrahydroxytrans-stilbene (98.3% inhibition IC 50 ¼29.1 mM) showed more potent activity against the enzyme than 3,3 0 -dihydroxy-trans-stilbene. 3,3,4,4 0 -tetrahydroxy-trans-stilbene (IC 50 ¼29.1 mM) inhibited the tyrosinase activity almost completely. It is seen that an increase of the inhibitory power of the hydroxystilbenes is correlated with an increase of the number of hydroxyl groups. O-methylation decreased the action of the stilbenes. the aglycone of which is resveratrol. The inhibitory activity of piceid was 6.9 and 8.2 (L-DOPA and L-tyrosine) times lower than that of resveratrol (phenylthiourea was used as a positive control).
The tested compounds showed a more significant effect on the monophenolase activity than on the diphenolase activity 128       Inhibitory effect for compounds A (IC 50 ¼6.71 mM) and B (IC 50 ¼14.7 mM) was higher than for kojic acid (IC 50 ¼28.9 mM), when L-tyrosine was used as a substrate. Inhibitory effect of compound B (IC 50 ¼82.3 mM) was significantly less than that of kojic acid (IC 50 ¼23 mM),when L-DOPA was used as a substrate. In the case of compound A the IC 50 value was comparable to that of kojic acid (IC 50 ¼24.6 mM) 130 .
The obtained results indicate that the deglycosylation of stilbenes has a positive influence on their activity and indicates that the aglycones are more active. This is probably related to particle size because as larger compounds, glycosides have restricted an access to the active site of the enzyme.

Isoprenyl chain.
The inhibition of tyrosinase may also be stimulated by the compounds with isoprenyl chain in their structure, e.g. 4-[(2 00 E)-7 00 -hydroxy-3 0 ',7 00 -dimethyloct-2 00 -enyl] À 2 0 ,3,4 0 ,5tetrahydroxy-trans-stilbene (compound A) and chlorophorin, both isolated from Chlorophora excels (Welw.) Benth. core, showed a different inhibitory activity dependent on the presence of the isoprenyl chain. The IC 50 values for compound A and chlorophorin were equal 96 and 1.3 mM, respectively (kojic acid, IC 50 ¼20 mM). Attaching a water molecule to the geranyl chain in compound A reduced its tyrosinase activity. This is probably due to reducing the chain's interaction and the hydrophobic protein pocket close to the active site 131 . The other investigations have shown that the presence of a prenyl chain in a compound having a 4-substituted resorcinol backbone increased the inhibitory activity compared to that of oxyresveratrol (IC 50 ¼0.66 and 0.98 mM, respectively).
In another study, the impact of chain length, functional groups (polarity), and cyclisation of isoprenyl chains on the anti-tyrosinase activity was investigated. An increase in chain length from one isoprene unit to two resulted in a 4-fold increase in activity (IC50 ¼ 15.87 mM, IC50 ¼ 60.14 mM). In turn, the introduction of hydroxyl groups or cyclisation of the isoprenyl chain caused a drastic decrease in anti-tyrosinase activity. Similar results of isoprenyl chain influence were observed in stilbene derivatives isolated from Angelica keiskei roots.

Double bond.
Another compound inhibiting tyrosinase is gnetol, a tetrahydroxystilbene isolated from Gnetum gnemon L. It was appeared that gnetol is approximately 30 times more potent than kojic acid. Additionally, gnetol inhibited tyrosinase much more than dihydrognetol (100% and 20%, respectively). The double bond is crucial for the activity. The double bond's role in the stilbene backbone in inhibiting tyrosinase examined the cis-olefin structure. The cis isomer of 3,3 0 -dihydroxystilbene (% inhibition c. 1) compared to the trans isomer (26% inhibition) shows no inhibitory effect. The saturation of the double bond in the oxyresveratrol (IC 50 ¼0.98 mM) significantly reduces the activity (IC 50 ¼58 mM). However, dihydrooxyresveratrol showed eight times more inhibitory effect on the activity of fungal tyrosinase than oxyresveratrol (IC 50 ¼1.6 and 12.7 mM, respectively). The higher activity of dihydrooxyresveratrol, compared to oxyresveratrol, was probably due to its dibenzyl structure, which provided greater flexibility, and thus allowed for a more effective interaction of phenolic groups with the enzyme (Figure 19) 132 .
Similar results of the effect of double bond saturation were obtained by studying dihydrostilbene derivatives isolated from the 80% ethanolic extract of the Dendrobium loddigesii Rolfe stem. It was appeared that 3,4,5-trihydroxy-3 0 ,4 0 -dihyroxyhydrostilbene; 3,5-dihydroxy-3 0 ,4 0 -dihyroxyhydrostilbene and their methoxy derivatives did not inhibit tyrosinase activity. The exception to this rule is 3,5-dihydroxy-3 0 -dihyroxyhydrostilbene (IC 50  that methylation of the 3-OH group inactivates the compound. Kojic acid was used as a positive control (IC 50 ¼8.02 mM). A noteworthy compound for (Q)SAR studies is 1,3-benzodioxol derivative (benzene ring linked to dioxolane). Due to the ambiguity of the results, further studies on the effect of a double bond in stilbenes are needed.   Table 16. An anti-tyrosinase activity of flavonoids (UE-unable to establish; c-with respect to L-tyrosine; d-with respect to both L-tyrosine and L-DOPA; NR-not reported; NT-not tested).   at a concentration of 100 mM inhibited the activity of tyrosinase at the level of 98%. However, the oligomers have appeared to be weak inhibitors, e.g. dimer-e-viniferin at a concentration of 100 mM showed approx. 24.6% inhibition, the trimmers, and tetrameters showed an inhibition level of less than 8%. It may be suggested that the inhibitory potency of the resveratrol oligomers decreases with increasing molecular weight ( Figure 20; Tables 4S and  Table 19) 133 .

Chalcones
Chalcones belong to the group of unsaturated aromatic ketones and are thought to be the precursors for the synthesis of flavonoids. The core of chalcones consists of two aromatic rings linked by a three-carbon a, b-unsaturated carbonyl system (1,3-diphenyl-2-propen-1-one) ( Figure 17). Chalcones can exist in trans (E) and cis (Z) forms, but cis isomers are unstable due to spherical effects. Chalcones are characterised by a broad spectrum of biological properties, including anticancer, antioxidant, antidiabetic, antiinflammatory, antibacterial, and antiviral activities 133 .
Chalcones have shown an anti-tyrosinase activity dependent on their structure and concentration. Khatib et al. studied the effect of catechol and resorcinol groupings in the A and B rings of chalcones. Two substrates for tyrosinase, L-tyrosine (first step -Ltyrosine to L-DOPA) and L-DOPA (second step -L-DOPA to oquinone), were used. All of the compounds tested showed greater activity in blocking the first step than the second step. 3,4,2 0 ,4 0 -Hydroxychalcone, with a resorcinol moiety (at the 2 0 and 4 0 positions) in the A ring and catechol in the B ring inhibited the first step more potently (IC 50 ¼29.3 mM) than the second step (IC 50 >100 mM). 2,4,3 0 ,4 0 -Hydroxychalcone, with the opposite structure to 3,4,2 0 ,4 0 -hydroxychalcone, inhibited tyrosinase about 146.5-fold more potently (IC 50 ¼0.2 mMstage 1, and IC 50 ¼7.5 mM stage 2). The compound 3,5,2 0 ,4 0 -hydroxychalcone, in which the OH groups of the B ring are located at positions 3 and 5 while maintaining the identical position of the catechol group as in 3,4,2 0 ,4 0 -hydroxychalcone, blocked the enzyme weaker (IC 50 ¼31.7 mM for step 1, IC 50 >1000 mM for step 2). Compound 2,4,2 0 ,4 0 -hydroxychalcone, made up of two resorcinol groups in rings A and B, blocked tyrosinase activity most strongly (IC 50 ¼0.02 mM for stage 1 and up to 90 mM for Stage 2). Additionally, 2,4,3 0 ,4 0 -hydroxychalcone is 7.5 times more active than trans-stilbene, with the same catechol and resorcinol arrangement. Moreover, the position of OH groups in the A and B ring affects the mechanism of chalcone inhibition. Compounds with a catechol group showed the ability to chelate copper ions, while 2,4,2 0 ,4 0 -hydroxychalcone (resorcinol structure) did not chelate copper ions. The catechol group in ring A acted as a chelator of copper ions, while the catechol in ring B is oxidised to o-quinone. However, catechol groups in the A or B ring had no significant impact on tyrosinase inhibition. The compound with two resorcinol moieties has the most potent effect on tyrosinase activity 134 .
Nguyen et al. provided more information about the influence of the position and number of OH groups on the activity of chalcones. It was appeared that a presence one or two hydroxyl groups in the A ring do not strength the inhibition, e.g. inhibition for 4-hydroxychalcone, 2-hydroxychalcone, and 2,4-dihydroxychalcone was about 14% at 50 mM, respectively. However, the changes had been observed after the introduction of the 4 0 -OH group in the B ring, which resulted in the activity's increase. The inhibition at 50 mM for 4 0 -hydroxychalcone, 2,4,4 0 -trihydroxychalcone was 71% and 67%, respectively. Additionally, the presence of the 2-OH group in the A ring drastically reduces the activity of 2,4 0 -dihydroxychalcone (10% inhibition at 50 mM concentration). The weakening effect of the 4 0 -OH group on tyrosinase activity is due to conformational changes. These changes are due to the formation of hydrogen bonds between 2-OH and the carbonyl group. The importance of the arrangement of hydroxyl groups is related to the structure of chalcones. Ring A is associated with a carbonyl carbon atom, while ring B is related to a vinyl carbon atom. This   is in contrast to stilbenes, in which the rings are bound to identical carbon atoms (Figure 21) 136 .
The anti-tyrosinase properties have also been confirmed in the group of the chalcone glycosides, e.g. licuraside, isoliquiritin, and the aglycone licochalcone A isolated from two species of Glycyrrhiza (Glycyrrhiza uralensis Fisch and Glycyrrhiza inflate Bat., respectively). The IC 50 for licuraside, isoliquiritin, and licochalcone A, in the presence of L-tyrosine, were of 72, 38, and 25.8 mM, respectively. The compounds inhibited enzyme competitive (Ltyrosine). The inhibitory effect of chalcones on diphenolase activity (L-DOPA as substrate) was much lower. The difference in inhibition between monophenolase and diphenolase activity is related to the structure of the chalcones, those ones which inhibit monophenolase more strongly show a similarity to L-tyrosine ( Figure 22) 134 .
The 4-OH group in the B ring of chalcones affects the potency of the inhibitor (similarity to the tyrosine backbone). Licochalcone A is a more potent inhibitor than licurad and isoliquiritin because it has a free 4-OH group in the B ring and has less steric hindrance. The sugar residue at the 4 0 -OH position hinders access to the enzyme's active centre, resulting in reduced inhibitory activity. In addition, the 3,3-dimethylpropylene group at position 5 (ring B) of licochalcone A disrupts the quaternary structure of tyrosinase, inhibiting the enzyme 134 .
In the case of diphenolase activity, for which the substrate is L-DOPA, the presence of both 3 0 -OH and 4 0 -OH groups in the B ring is necessary in the inhibitor's structure in order to resemble L-DOPA. The absence of the 3 0 -OH group in the B ring of chalcones resulted in the lack of diphenolase inhibitory activity of tyrosinase 137 . The tyrosinase activity may be also regulated via the introduction of alkyl chains into chalcone molecules. It has been proved that prenylated chalcone, curaridine, blocked the enzyme activity very strongly (IC 50 ¼0.6 mM) compared to kojic acid (IC 50 ¼20.5 mM). The important elements of the studied compound are the 2 0 -OH and 4 0 -OH groups in the B ring, and 4-OH, and the prenyl chain at C-5 (lavandulyl) in the A ring 138 . On the other hand, the addition of two isoprenyl groups at the C-5 and C-3 0 positions to 4,4 0 ,6-trihydroxychalcone abolishes tyrosinase inhibition. This is probably due to steric hindrance (Figure 23; Tables  5S and Table 20) 134 .
Summarising results of the above studies, they indicate the significance of the presence of a feruloyl group at C-6, an acetyl group at C-6 0 , and another acetyl group at C-3 0 /C-4 0 in inhibiting of the enzyme. Additionally, it seems that the introduction of additional feruloyl groups in the fructose moiety increases the activity of the compounds (Table 21).

Coumarin
Coumarins are derivatives of a-pyrone, condensed with benzene. Benzo-a-pyrone is usually substituted at C-7, less often at C-5, C-6, and C-8 positions with a hydroxyl group to which methyl groups

Presence of double bond in trans position increases activity of stilbenes
The increase in the number of hydroxyl groups enhances the activity of stilbenes The presence of residual sugar reduces the activity of stilbenes The presence of isoprenyl chain increases the inhibitory activity of stilbenes The leading structure of stilbenes or sugar moieties may be attached. A furan or pyran ring may be condensed with the benzo-a-pyrone structure ( Figure 25). Coumarins isolated from Euphorbia lathyris seeds showed weak inhibitory properties against tyrosinase. The exception was esculetin, whose IC 50 was 43 mM (kojic acid; IC 50 ¼10 mM) l 144 . Different results were obtained for the constituents present in the leaf extract from M. alba L. Scopoletin showed the strongest anti-tyrosinase properties (IC 50 ¼0.2 mM), while esculetin and scopoline appeared to be less potent with the IC 50 equal 6.9 and 15.9 mM, respectively. All inhibitors blocked the enzyme competently 145 .
Coumarin glycosides present in the Morus nigra roots showed weak inhibitory properties against tyrosinase, probably the presence of a sugar residue reduced the inhibitory activity 146 .
More information on the structure-activity relationship was provided by examining semi-synthetic/synthetic coumarin derivatives. In a study conducted by Matos et al., the effect of 3-phenylcoumarin and 3-thiophenylcoumarin derivatives on tyrosinase activity was investigated. L-DOPA was used as a substrate for the enzyme. The results showed that some synthesised derivatives exhibited an inhibitory activity against mashroom tyrosinase. The two most active compounds (5,7-dihydroxy-3-(3-thiophenyl)coumarin and 3-(4 0 -bromophenyl)-5,7-dihydroxycoumarin) showed tyrosinase inhibitory activity (IC 50 ¼0.19 and 1.05 mM, respectively), higher than kojic acid (IC 50 ¼17.90 mM). The presence of two hydroxyl groups at the C-5 and C-7 positions of the coumarin scaffold improved the inhibitory activity. The presence of the resorcinol grouping enhanced the ability to chelate copper ions 148 .
In subsequent studies, the effect of the position of hydroxyl, methoxyl, ethoxyl, and bromine groups in 3-phenylcoumarin derivatives on the activity against tyrosinase was examined. 3-Phenyl-6-hydroxy-8-bromocoumarin (IC 50 ¼215 mM) inhibited tyrosinase more strongly than kojic acid (IC 50 ¼420 mM). In addition, the introduction of more hydroxyl groups in the coumarin grouping increased the inhibitory activity. Compared to 6-hydroxy-8bromocoumarin (IC 50 ¼302 mM), one more hydroxyl group was introduced in the 3-phenyl-6-hydroxy-8-bromocoumarin, which   Pinostilben ( improved the inhibitory activity about 1.5 times. A bromine substituent and a C-4 0 hydroxyl group at the C-6 positions increase the inhibitory activity. In turn, methoxy and ethoxy derivatives weakly inhibited the enzyme. It seems that brominated hydroxycoumarin derivatives may be promising inhibitors of tyrosinase 149 .
In the study of Asthan et al., the effect of the position of the hydroxyl group in benzo-a-pyrone on tyrosinase activity was checked. The position of the hydroxyl group at C-6 and C-7 causes the molecule to behave as a weak substrate for the enzyme. This is related to the interaction of the hydroxyl group with the copper ion in the enzyme's active centre, which means that the compounds with the OH group in the pyrone ring cannot be substrates for tyrosinase. Among investigated compounds, only 3-hydroxycoumarin inhibited the enzyme activity. The studies indicate the possibility of application of 3-hydroxycoumarin structure as a new class of tyrosinase inhibitors 150 .
Another study examined the effect of tannins isolated from the methanolic extract of Ecklonia stolonifera. The extract contained five phlorotannins: phloroglucinol, eckstolonol, eckol, phlorofucofuroeckol A, and dieckol. Particularly noteworthy was dieckol (IC 50 ¼2.16 mg/mL), which blocked the enzyme more strongly than kojic acid (IC 50 ¼6.32 mg/mL) and arbutin (IC 50 ¼112.0 mg/mL). Phloroglucinol and eckstolonol showed the competent type of inhibition. Eckol, phlorofucofuroeckol A, and dieckol inhibited the enzyme incompetently. The isolated phlorotannin derivatives owe       their properties to the presence of a resorcinol group, which can chelate copper in the active tyrosinase center 157 . Shoji et al. examined the effect of procyanidin polymerisation on tyrosinase activity. All oligomer fractions (1mer to 7mer) strongly inhibited tyrosinase activity, as did kojic acid. The IC 50 values were similar for all groups: monomer, 74 mM; dimer, 235 mM; trimmer, 140 mM; tetramer, 149 mM; pentamer, 184 mM; hexamer, 127 mM; and heptamer 103 mM. No correlation was observed between the degree of procyanidin polymerisation and tyrosinase inhibition. Nevertheless, these observations suggest that procyanidins are effective inhibitors of tyrosinase (Table 23) 158 .

Conclusion and future perspectives
Despite the fact that the plant-derived substances have always been a mainstay in medical treatment, the use of plants as a source of new drugs is still poorly studied. Of the 250,000-500,000 plant species, only a small number have been adequately studied. Besides the possibility of a direct use of plant materials in medicine, isolated substances can be used as a source of the structures for the synthesis of new drugs.
Commercially available Hyal and tyrosinase inhibitors are characterised by a range of adverse properties. Tyrosinase overactivity can be reduced by inhibitors such as hydroquinone, arbutin, vitamin C, azelaic acid, kojic acid, and ellagic acid. However, those inhibitors have many side effects, e.g. hydroquinone, the most commonly used inhibitor, shows mutagenic and irritating effects (dermatitis and irritation), while arbutin, a prodrug of hydroquinone, is a chemically unstable compound. Kojic acid is carcinogenic, L-AA is quickly degraded, ellagic acid, due to its poor solubility, shows a low bioavailability. Therefore, it is necessary to search for new safe inhibitors [161][162][163][164][165] .
One of the Hyal inhibitors available for treatment is escin (aescin). Unfortunately, due to its physicochemical properties, the compound is absorbed orally only to a small extent 166 .
From this review, it is clear that currently used tyrosinase and Hyal inhibitors are not without their drawbacks, therefore, there is a great need to search for the new inhibitors with more valuable pharmacological properties. In the further bioprospecting, the following criteria should be considered: Agarozizanol F -Yang et al. 159 1. Examine the effect of extracts and newly isolated compounds on cell cultures to determine cytotoxicity. Some compounds, e.g. alkaloids or terpenes, may exhibit toxic effects. Therefore, early determination of their toxicity is necessary. In addition, the study of new inhibitors using cell cultures will allow us to initially determine the metabolic pathways and mode of absorption (active or passive transport) of the tested compounds. 2. To better understand the utility of new substances in inhibiting Hyal or tyrosinase, more studies using animal models should be planned to determine the safety and efficacy of the tested inhibitors. 3. Most of the work was done with tyrosinase isolated from the fungus Agaricus bisporus. Human tyrosinase is membranebound, while fungal tyrosinase is a cytosolic enzyme. Furthermore, the human enzyme is a monomer that undergoes an intensive glycation process, unlike the fungal tyrosinase, which is a tetramer. Knowledge about the effects of tyrosinase inhibitors on the human enzyme is limited, thus, it is necessary to determine the suitability of fungal tyrosinase inhibitors relative to the human enzyme. 4. The search for selective Hyal inhibitors is also essential. The availability of selective inhibitors that target one type of Hyal without affecting another Hyal is important because different isoforms of Hyal regulate physiological processes. Additionally, the search for selective Hyal inhibitors will allow for a better understanding of the role of Hyals in both physiological and pathological processes. 5. A significant problem when comparing the results of studies is the deficiencies in analysing enzymatic reaction kinetic. Information on Km, Vmax, and Cmax of the enzymatic reaction is often missing in studies. 6. Many studies, especially on Hyal, focus only on determining the properties of anti-Hyal extracts. A more careful analysis of the active ingredients is needed.